This chapter combines the current laboratory diagnostic methods for measles virus and rubella virus for convenient review and reference. The measles virus genome is 15,894 nucleotides in length and contains six structural genes organized on the single strand of RNA in a gene order consistent with those of most of the paramyxoviruses, i.e., 3'-N, P. M, F, H, L-5'. A recent review by Rota et al. provides an excellent overview of the current status of the molecular epidemiology of measles and the global distribution of the various genotypes. The most common complications associated with measles virus infection are otitis media (7 to 9%), pneumonia (1 to 6%), and diarrhea (6%). Suitable samples for isolation of measles virus or for detection of viral antigen can be whole blood, serum, throat and nasopharyngeal secretions, urine, and, in special circumstances, brain and skin biopsy samples. Characteristic cytopathic effects (CPE) of measles virus infection include multinucleated cells and cellular inclusions (in-tracytoplasmic and intranuclear). The reverse transcriptase PCR (RT-PCR) should be considered for diagnostic use where IgM testing is compromised by the concurrent or recent use of measles virus-containing vaccine as part of an outbreak response or in settings of recent vaccine distribution, such as supplemental immunization activities. Groups of related viruses within the clades have been classified as genotypes. Time course of rubella virus-specific IgM and IgG detection by enzyme-linked immunosorbent assays (ELISAs) in sera of rubella patients.

CPE of measles virus when propagated in Vero/hSLAM cells, a cell line that is transfected with a gene encoding the wild-type virus receptor (CDw150) for measles virus. The cell line naturally expresses the simian form of CD46 on the cell surface. (A) Uninfected monolayer of Vero/hSLAM cells; (B) measles virus-infected monolayer of Vero/hSLAM cells with apparent syncytium formation and multinucleated giant cells (arrows).

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FIGURE 1

CPE of measles virus when propagated in Vero/hSLAM cells, a cell line that is transfected with a gene encoding the wild-type virus receptor (CDw150) for measles virus. The cell line naturally expresses the simian form of CD46 on the cell surface. (A) Uninfected monolayer of Vero/hSLAM cells; (B) measles virus-infected monolayer of Vero/hSLAM cells with apparent syncytium formation and multinucleated giant cells (arrows).

IFA of rubella virus-infected cells using monoclonal antibody to the E2 protein. Vero cells were infected with wild-type rubella virus and 3 days after infection were fixed with cold paraformaldehyde, followed by permeabilization with -20°C methanol. Reaction of infected cells with monoclonal antibody to the E2 protein (monoclonal antibody 24–26; Meridian Life Science, Saco, ME) was followed by detection of bound monoclonal antibody with Alexa Fluor 488-conjugated, highly cross-absorbed goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR). Cell nuclei were visualized by staining with propidium iodide. About 25% of the cells which stained with propidium iodide were positive for E2 protein.

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FIGURE 2

IFA of rubella virus-infected cells using monoclonal antibody to the E2 protein. Vero cells were infected with wild-type rubella virus and 3 days after infection were fixed with cold paraformaldehyde, followed by permeabilization with -20°C methanol. Reaction of infected cells with monoclonal antibody to the E2 protein (monoclonal antibody 24–26; Meridian Life Science, Saco, ME) was followed by detection of bound monoclonal antibody with Alexa Fluor 488-conjugated, highly cross-absorbed goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR). Cell nuclei were visualized by staining with propidium iodide. About 25% of the cells which stained with propidium iodide were positive for E2 protein.

Time course of rubella virus-specific IgM and IgG detection by ELISA in sera of rubella patients. Commercial IgM capture ELISA (A) and IgG indirect ELISA (B) were used to detect rubella virus-specific antibodies at the indicated number of days after onset of symptoms (usually rash); antibody index and ISR are the commercial test designations for the ratio of the optical density obtained for the test serum to the optical density obtained for a standard (cutoff) serum. The minimum signal considered positive in each test is indicated by a dashed line. Only results from patients who tested positive for IgM to rubella virus at some time after the onset of symptoms are shown.

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FIGURE 3

Time course of rubella virus-specific IgM and IgG detection by ELISA in sera of rubella patients. Commercial IgM capture ELISA (A) and IgG indirect ELISA (B) were used to detect rubella virus-specific antibodies at the indicated number of days after onset of symptoms (usually rash); antibody index and ISR are the commercial test designations for the ratio of the optical density obtained for the test serum to the optical density obtained for a standard (cutoff) serum. The minimum signal considered positive in each test is indicated by a dashed line. Only results from patients who tested positive for IgM to rubella virus at some time after the onset of symptoms are shown.

23.Centers for Disease Control and Prevention. 2008. Recommendations from an ad hoc meeting of the WHO measles and rubella laboratory network (LabNet) on use of alternative diagnostic samples for measles and rubella surveillance. MMWR Morb. Mortal. Wkly. Rep.57: 657– 660.

31.Cohen, B. J.,, S.Audet,, N.Andrews,, J.Beeler, and on Behalf of the WHO Working Group on Measles Plaque Reduction Neutralization Test. 2007. Plaque reduction neutralization test for measles antibodies: description of a standardised laboratory method for use in immunogenicity studies of aerosol vaccination. Vaccine26:59–66.

101.Reef, S. E.,, T. K.Frey,, K.Theall,, E.Abernathy,, C. L.Burnett,, J.Icenogle,, M. M.McCauley,, and M.Wharton. 2002. The changing epidemiology of rubella in the 1990s: on the verge of elimination and new challenges for control and prevention. JAMA287:464–472.

a Times and percentages given are approximate and are meant to guide typical specimen collection. Percentages vary depending on the sensitivity of the assay used. Note that the times listed in the third column were chosen to help guide specimen collection and may not be the earliest time when >90% of cases are positive.

b After maximum number of cases are positive for a given criterion, the approximate time for 50% of cases to become negative.

c “Alternative” specimens, OMT and DBS, have been evaluated for detection of virus (OMT) and IgM (OMT and DBS). See references 1, 23, 55, 69, and 98.

f Declining maternal IgG and developing IgG response in a CRS patient lead to high (steady) or increasing IgG levels in the CRS patient through the first year of life.

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TABLE 2

Timing of biological markers of rubella virus infectiona

a Times and percentages given are approximate and are meant to guide typical specimen collection. Percentages vary depending on the sensitivity of the assay used. Note that the times listed in the third column were chosen to help guide specimen collection and may not be the earliest time when >90% of cases are positive.

b After maximum number of cases are positive for a given criterion, the approximate time for 50% of cases to become negative.

c “Alternative” specimens, OMT and DBS, have been evaluated for detection of virus (OMT) and IgM (OMT and DBS). See references 1, 23, 55, 69, and 98.